Genomic instability is a hallmark of aging cells, and the major cause of tumorigenesis. The long-term goal of the proposed research is to understand the molecular mechanism of age-related genomic instability. Genomic rearrangements are thought to arise as a result of errors in the repair of DNA double-strand breaks (DSB). We have recently developed a sensitive fluorescent assay for analysis of DNA DSB repair via nonhomologous end joining (NHEJ) pathway in senescent cells. Using this assay we have demonstrated that NHEJ becomes less efficient and more error-prone during cellular senescence. Our preliminary data suggest that the protein levels of several key components of NHEJ pathway are reduced dramatically in senescent cells, consistent with the observed age-related decline of NHEJ. Therefore, the focus of this application is to further examine NHEJ during aging and to understand the mechanism responsible for the low levels of NHEJ proteins in senescent cells. Our Specific Aims are: (1) To test the hypothesis that NHEJ becomes less efficient and more error-prone during organismal aging. General decline of DNA repair has been observed in aged individuals, suggesting that NHEJ is likely to decline during aging. We will examine the efficiency and fidelity of NHEJ in fibroblasts from donors of different age from the Baltimore Longitudinal Study on Aging, and in aging mice. We will use the NHEJ assay we developed for analysis of end joining in senescent cells. (2) To test the hypothesis that the decline of NHEJ is caused by reduction in the amount of DSB repair proteins. Our preliminary experiments show that Ku70, Ku80 and NBS1 proteins are virtually absent from senescent cells. To expand this observation, we will examine mRNA and protein levels of the key components of NHEJ machinery in replicatively senescent human fibroblasts and in tissues of old mice. We will determine whether a transcriptional or post-translational mechanism is responsible for the reduction of NHEJ protein levels: (3) Generate a transgenic mouse model for analysis of age-related changes in NHEJ. Human genome becomes unstable with age leading to cancer and age-related diseases. Why genomes become unstable with age is unknown. The proposed study aims to understand the mechanism of age-related genomic instability, which will help develop ways to stabilize the aging genome and prevent cancer.